We propose a combination biochemical and cellular experiments to test hypotheses about both the molecular mechanisms and biological functions of 3 of the most abundant actin binding proteins. The biochemical and biophysical analysis of reaction mechanisms will provide definitive quantitative models for how the components interact. This information will complement our efforts to determine the atomic structures of these protein on another NIH grant. We will also test the functions of these proteins in live cells. Our long range goal is not only to provide insights about these particular proteins, but also to identify some general principles about the function of the actin cytoskeleton. 1. Profilin is the classic actin monomer binding protein and may also participate in phosphoinositide signalling. We will examine the functions of profilin in yeast and vertebrate cells using depletion of endogenous profilins and expression of naturally occurring variants of profilin. From biochemical and biophysical studies we will work out the mechanism by which profilin catalyzes the nucleotide exchange of actin monomers and the complex of actin with thymosin beta4. We will identify the residues in profilin-II responsible for its high affinity of PIP2 and then use kinetic analysis to determine the mechanism of binding of profilins to PIP2 in neutral lipid bilayers. We will use binding and enzyme assays to learn how profilin and tyrosine phosphorylation of phospholipase C-1 regulate its activity in the PI signalling pathway. To explore the hypothesis that profilin itself might be a second messenger during phosphoinositide signalling, we will characterize the dissociation of profilin from bilayers containing PIP2. We will also examine the effects of profilin on PIP3-and 4-kinase activity, characterize interactions of profilin with yeast cyclase associated protein and characterize the protein components of an Acanthamoeba profilin-binding complex. 2. Actophorin represents a ubiquitous class of small actin filament severing proteins. We will identify contact sites between actin and actophorin by chemical crosslinking, measure the affinity of actophorin for actin monomers and actin filaments and use fluorescent microscopy to characterize actin filament severing by actophorin. We will use the atomic model for the complex of actin and actophorin to design and characterize mutants of actophorin for testing physiological functions in yeast. 3. Alpha-actinin is the classic actin filament crosslinking protein and contains an actin binding site similar to several other proteins. We will measure the kinetic constants for the binding of alpha-actinin to actin filaments and use these parameters as the basis for mathematical models to explain the observed mechanical properties of gels of actin filaments and alpha-actinin. We will measure the binding properties of other members of the alpha-actinin head family and use them to predict the dynamic mechanical properties of related structures such as the membrane cytoskeleton. We will also attempt to crystallize the actin binding domain of alpha-actinin for future structural studies.